The present invention relates to a novel stabilizing agent for live viruses, for use of the latter as vaccines and to the stabilized vaccines so-obtained.
Vaccination by inactivated antigens or attenuated live virus strains, have long been used to protect men and animals against viral diseases. In both cases, it is imperative for the integrity of the viral antigens to be preserved in the vaccines in order that the vaccinating effect of the latter may be effective after preparation, storage and transportation of the vaccines to the places of vaccination. Now live attenuated vaccines are particularly fragile and their vaccinating action is rapidly destroyed when they are exposed to temperatures above +4.degree. C. and sometimes even below. Such instability of the vaccines is unacceptable, particularly when these vaccines are used, like anti-yellow fever vaccine, on a large scale in tropical countries where they may accidentally be exposed to high ambient temperatures which are prevailing in these regions, before being administered, considering that the maintenance of the stability of the titer of the live vaccines is the primordial factor for ensuring effective immunization of the vaccinated populations, particularly in regions with a hot climate, and especially in cases where the maintenance of a cold storage line from the manufacturing laboratory to the places of utilization may prove to be insufficient or may be interrupted. It is therefore necessary to be able to provide a stable and heat-resistant vaccine, capable of withstanding deterioration when it is stored for long periods and is exposed accidentally to high temperatures. To confer on live vaccines the heat stability indispensable for the maintenance of the entireness of their vaccinating power or infectivity, it has been proposed to prepare these vaccines, particularly vaccines against measles, rubella, mumps, and yellow fever, in lyophilized form. However, even in this stabilized form, these lyophilized live vaccines are still very fragile, to the point that they must be stored at -30.degree. C. and are shipped in solid carbon dioxide (known under the trademark "CARBOGLACE") at -80.degree. C. However, even by complying with these extremely drastic restrictions of storage and shipment, it is not always possible to avoid the vaccines being exposed, in the course of their storage and their shipment, to high temperatures which causes loss of infectivity of these vaccines and hence, their loss of activity, so that the vaccinated populations are not in fact, protected in any way by vaccination by a vaccine which has lost its infectivity. This loss of infectivity (or titer) under the effect of a rise of temperature, has been clearly shown in a study published by the World Health Organisation, 1974, 44,729-737 under the signature of Y. Robin, A. C. Saenz, A. S. Outschoorn and B. Grab, entitled "Study of the heat stability of anti-yellow fever vaccine in samples of eight batches coming from various countries" whence it emerges that vaccinating compositions stabilized by lyophilization lose about 75% of their initial infectivity following storage for 6 months at +5.degree. C. for two weeks at +22.degree. C., and about 95% of their initial infectivity after one week at 37.degree. C. This is the reason why it has been attempted to improve the heat stability of lyophilized live vaccines, by adding stabilizers thereto. Thus, in French Pat. No. 1,548,489 and Special Medicament Patent 7321M, the Recherche et Industrie Therapeutiques R.I.T. Company proposed adding to attenuated live rubella virus, before or after lyophilization, a stabilizing agent constituted by Hanks solution supplemented with casein hydrolysate (if necessary completed with a second stabilizing aqueous solution containing potassium glutamate, saccharose and chloramphenicol). Results relating to the heat stability of vaccines against measles, lyophilized and reconstituted have, moreover, been made public by this company at the 15th Congress of the AISB on vaccinations in underdeveloped countries, held at Guadeloupe in 1978, and published in Develop. biol. Standard. vol. 41, p. 259-264 (S. Karger Ed., Bale, 1978): the experiments reported in this publication show that with the stabilizing agent recommended by the R.I.T. company, the titer loss is 0.30 log.sub.10 after two days and 0.70 log.sub.10 after one week, for a lyophilized vaccine exposed to a temperature of 41.degree. C. on a water bath for 14 days, then reconstituted with diluant preheated at 41.degree. C., without the reconstitution causing an additional decrease in the titer. It is to be noted that the Hanks solution recommended according to the R.I.T. Patents, is an aqueous solution containing inorganic salts, glucose and phenol red and that the casein hydrolysate is essentially constituted by the following amino acids: glutamic acid, leucine, proline, lysine, valine, tyrosine, methionine, phenylalanine, arginine, and histidine. It has also been proposed (cf. French Pat. No. 2,371,927 in the name of Connaught Laboratories Ltd.) to stabilize a vaccine against poliomyelitis, with a stabilizing aqueous composition containing about 0.3-1.0M of Tris (hydroxy-)aminomethane buffer, about 100 .mu.g/ml of L-cystine and about 0.8 mg/ml of vaccine, of gelatin hydrolysed by an acid, the hydrolysed gelatin being essentially constituted by the following amino acids: glycocoll, alanine, leucine, phenylalanine, tyrosine, serine, proline, hydroxyproline, aspartic acid, glutamic acid, arginine, lysine, tryptophane. French Patent to Merck & Co. No. 2,424,031 describes, as for it, a "vaccine stabilizer" recommended for stabilizing a vaccinating composition in the lyophilized or liquid state (such as virus of measles, of mumps, of rubella, of chicken pox, of poliomyelitis, of hepatitis, of herpes simplex 1 or 2, or of combinations of these viruses) by means of a stabilizing aqueous solution containing hydrolyzed gelatin, a polyalcohol such as sorbitol and a sufficient amount of acid buffer to maintain the pH between about 6.0 and 6.5, which buffer can be phosphate, acetate or citrate buffer. In the "Journal of Biological Standardization" ( 1980), 8, p. 281-287, W. J. McAleer, H. Z. Markus, A. A. McLean, E. B. Buynak and M. R. Hilleman have reported experiments that they carried out to determine the stability on storage at various temperatures, of live vaccines against measles, mumps and rubella suspended in this novel stabilizing agent. They have shown that the antimeasles lyophilized vaccine loses less than one third of its infectivity when it has been stored for two months at 36.degree.-38.degree. or for one month at 44.degree.-46.degree. C.